This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Corticotropin-releasing factor type 2 receptors (CRFR2) are suggested to facilitate
successful recovery from stress to maintain mental health. They are abundant in the
midbrain raphe nuclei, where they regulate serotonergic neuronal activity and have
been demonstrated to mediate behavioural consequences of stress. Here, we describe
behavioural and serotonergic responses consistent with maladaptive recovery from stressful
challenge in CRFR2-null mice.

Results

CRFR2-null mice showed similar anxiety levels to control mice before and immediately
after acute restraint stress, and also after cessation of chronic stress. However,
they showed increased anxiety by 24 hours after restraint, whether or not they had
been chronically stressed.

Serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) contents were quantified
and the level of 5-HIAA in the caudal dorsal raphe nucleus (DRN) was increased under
basal conditions in CRFR2-null mice, indicating increased 5-HT turnover. Twenty-four
hours following restraint, 5-HIAA was decreased only in CRFR2-null mice, suggesting
that they had not fully recovered from the challenge. In efferent limbic structures,
CRFR2-null mice showed lower levels of basal 5-HT in the lateral septum and subiculum,
and again showed a differential response to restraint stress from controls.

Conclusions

These results suggest that CRFR2 are required for proper functionality of 5-HT1A receptors in the raphe nuclei, and are key to successful recovery from stress. This
disrupted serotonergic function in CRFR2-null mice likely contributes to their stress-sensitive
phenotype. The 5-HT content in lateral septum and subiculum was notably altered. These
areas are important for anxiety, and are also implicated in reward and the pathophysiology
of addiction. The role of CRFR2 in stress-related psychopathologies deserves further
consideration.

Keywords:

Background

Serotonin (5-HT) is a key neurotransmitter in the control of mood. It is the major
target of current antidepressant medications, and often also of treatments for anxiety
disorders [1,2]. The principal sources of 5-HT neurons projecting to the forebrain are the midbrain
dorsal (DRN) and median (MRN) raphe nuclei [3,4].

Corticotropin-releasing factor (CRF) is a key mediator of the stress response [5-7], and anxiety and affective disorders have been associated with CRF hyperactivity
[8]. Corticotropin-releasing factor receptors are abundant in both DRN and MRN [9-11], where they are expressed in serotonergic and non-serotonergic neurons, including
regulatory GABAergic neurons [12,13], suggesting the potential for complex interactions between CRF and serotonergic systems.
Electrophysiological studies show that exogenous CRF administered to the raphe modulates
serotonergic neuronal firing activity [14-16], and therefore CRF receptor-mediated effects on stress-related behaviours may be
mediated via 5-HT in vivo[17-20].

Type 1 (CRFR1) and type 2 (CRFR2) CRF receptors [21-23] are preferentially activated by CRF or urocortin neuropeptides (Ucn1, Ucn2, Ucn3),
respectively [24-28]. The raphe nuclei receive inputs from both CRF and Ucn1 expressing neurons [14,15,29-31], and a potentially important role for the CRF system in controlling 5-HT neurons
here is emerging.

CRFR2 is expressed at high levels in the raphe nuclei, while CRFR1 is expressed at
lower levels in the raphe nuclei in rats and appears to be absent from this area in
mice and human beings [9-11]. Exogenously administered CRFR2 agonists induce c-Fos expression in DRN 5-HT neurons,
increase their firing rate, and increase 5-HT release in efferent stress-related nuclei
[32-36]. In pharmacological studies, CRFR2 activation in the DRN potentiates immediate fear
responses [35], fear conditioning and escape deficits 24 hours later in a model of learned helplessness
[37,38], and decreases exploratory behaviours [19] in rodents. Recently, altered anxiety-like behaviour in Ucn-knockout or Ucn-overexpressing
mice has been linked to disturbances in serotonergic activity in the neural circuitry
controlling anxiety [39-41]. The Ucn1/Ucn2/Ucn3 triple knockout mouse phenotype suggests that CRFR2 and particularly
Ucn3 are involved in successful recovery from stress [41]. This interaction with the 5-HT system may provide a major link between the two main
arms of the central stress response; the CRF/Ucns peptidergic pathways and the sympathetic
monoaminergic system.

5-HT1A receptors (5-HT1AR) are also particularly associated with modulating anxiety [42] and pharmacological stimulation of CRF receptors in the raphe nuclei has been demonstrated
to regulate serotonergic neuronal firing here [43,44]. Thus, CRF-containing neuronal projections from the central amygdala (CeA) to the
raphe nuclei [45] may modulate activity at postsynaptic 5-HT1AR by directly regulating activity of efferent 5-HT projections or may have wider-ranging
effects on 5-HT function via altered raphe 5-HT1AR autoreceptor activity. Conversely, 5-HT1AR activity can influence CRF-induced changes in behaviour; 5-HT1AR-selective agonists can attenuate CRF-induced grooming [46]. We have previously shown that 5-HT1AR responsiveness plays a key role in stress-related behaviours associated with chronic
activation of CRFR2 [39] and that interaction is further explored in the studies presented here.

Activation of CRFR2 affects anxiety-like behaviour under stressed conditions [47-49] and CRFR2-null mice have an anxiogenic phenotype [50,51]. This raises the question of what role CRFR2 might play in the pathophysiology of
anxiety-related and affective disorders in human beings. To further investigate the
mechanisms underlying this, we examined the anxiety phenotype of CRFR2-null mice in
detail, and characterized their serotonergic responses to stress.

Methods

Animals

Mice were housed in temperature- and lighting-controlled rooms (lights on, 12 h) with
free access to laboratory chow and water. CRFR2-null mice, as previously described
[50], and control littermates (C57BL6 × 129) were the adult male offspring of parents
heterozygous for the knockout allele. For CRFR2 mRNA studies, adult male wild type
C57BL6/J mice (Harlan Laboratories) were used. Mice were group housed, except for
the chronic variable mild stress (CVMS) protocols, for which they were singly housed.
Principles of laboratory animal care (NIH No. 85-23, 1985) were followed. All procedures
were approved by The Weizmann Institute Animal Use and Care Committee or the United
Kingdom Animals (Scientific Procedures) Act, 1986.

Behavioural testing

Tests were carried out during the dark phase of the light cycle on adult male mice
(2 to 4 months). Mice were habituated in the home cage in a dark room for 2 hours
prior to each behavioural test. Separate groups of mice were tested under: (a) basal
conditions with no stress applied prior to testing, n = 12 for control group, n = 14 for CRFR2-null group; (b) immediately following 30 min of acute restraint stress
(ARS), n = 13, both groups; (c) 24 to 48 hours following ARS, n = 5 for control group, n = 8 for CRFR2-null group (light/dark transfer test performed at 24 hours post-stress,
open-field at 48 hours post-stress); (d) 3 to 4 days following a 4-week CVMS protocol,
(light/dark transfer test performed at 3 days post-stress, open-field at 4 days post-stress),
n = 10 for control group, n = 11 for CRFR2-null group. The mice of group d were then retested 3 weeks later,
when an ARS was applied and testing was performed at 24 to 48 hours. Figure 1 shows the timeline of the experimental protocols with stress procedures.

Figure 1.Schematic representation of experimental protocols and timelines. (A) Separate cohorts of CRFR2-null and control mice were tested for anxiety-like behaviour
in the light/dark transfer and open-field tests: under basal conditions; immediately
following ARS; following CVMS and again 24 to 48 h after an ARS applied 3 weeks after
the end of CVMS; 24 to 48 h following ARS. (B) CRFR2-null and control mice were exposed to no stress, ARS or CVMS, and mRNA expression
of stress-related genes and serotonin transporter (SERT) binding were quantified 12 h
after the end of stress. 5-HT/5HIAA content in brain nuclei were quantified in unstressed
mice and 24 h after ARS. (C) LCMRglu was measured in CRFR2-null and control mice one hour after administration
of saline or 5-HTR agonist. (D) CRFR2 mRNA levels in brain were quantified over a 48-h time course following ARS
or 7 days after CVMS in control mice. , ARS; , CVMS; d, days; w, weeks.

Open-field (OF) test

The apparatus and experimental conditions were as previously described [50]. Mice were placed in the centre of the apparatus to initiate a 10-min test session.
Visits to, and distance travelled and time spent in the inner zone of the arena were
quantified using a video tracking system (VideoMot2; TSE Systems, Bad Hamburg, Germany).

Light/dark transfer test (LDT)

Apparatus and experimental conditions were as previously described [50]. During a 5-min test session, visits to, and distance travelled and time spent in
the light compartment were measured.

Stress procedures

Mice were subjected to 30 min ARS in a ventilated 50 ml plastic centrifuge tube. The
CVMS regime was modified from Ducottet et al.[52]. Mice were singly housed and a variety of mild stressors were applied on an unpredictable
schedule, 2 to 3 stressors per day for 4 weeks; these included disruptions to the
light-dark cycle, cage shift to one previously inhabited by another male, cage tilt,
damp bedding, low-intensity stroboscopic illumination, white noise, restraint stress,
short periods of food or water restriction, and housing with no bedding followed by
water in the cage. Controls were housed under stress-free conditions.

In the CVMS paradigm, mice were behaviourally tested 48 hours following termination
of the last stressor, which was standardized and was 24 hours of constant light for
all mice (n = 10 or 11). For in-situ hybridization and 5-HT transporter (SERT) binding studies, mice (n = 6 for control basal group, n = 8 for CRFR2-null basal group, n = 7 for all stress groups) were killed 12 hours after ARS, or after the last variable
stressor, by decapitation within 15 s of disturbing the home cage. The brains were
removed, rapidly frozen on dry ice and stored at −80°C until analysis.

Analysis of tissue concentrations of 5-HT and 5-HIAA

Mice (n = 7 for unstressed groups, n = 6 for ARS groups) were killed by decapitation under basal conditions or 24 hours
following ARS. Brains were stored at −80°C until analysis. Areas selected for microdissection
were identified by comparison with a standard mouse brain stereotaxic atlas [56]. To ensure accuracy, we used a stereomicroscope to visualize neuroanatomical landmarks
for use as reference points in identifying specific nuclei and subdivisions of the
DRN. Small diameter microdissection tools (310 to 410 μm diameter) were used to restrict
dissections to the subregion of interest. High-pressure liquid chromatography analysis
of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) was performed, as previously described
[57].

CRFR2 mRNA qPCR analysis

Quantitative PCR for CRFR2 mRNA expression was carried out as previously reported
[40] in brain taken from naïve mice (controls), or 3, 6, 12, 24 or 48 hours after ARS,
or, for CVMS mice, one week after the end of the stress protocol (n = 8 all groups).

In-situ hybridization (ISH) histochemistry

Coronal brain sections (10 μm) were cut on a cryostat, thaw-mounted onto silanized
glass slides, and stored at −80°C until use. In-situ hybridization procedures and probes were as previously described [58-60]. Plasmids (generous gifts from Professor M. Holmes and Dr V. Bombail) containing
cDNA fragments for glucocorticoid receptor (GR), mineralocorticoid receptor (MR),
5-HT1A R, 5-HT2CR and tryptophan hydroxylase 2 (TPH2) were used to generate 35S-UTP-labelled specific antisense probes to mRNAs. Following ISH, slides were dipped
in Kodak Autoradiography Emulsion (Molecular Imaging Systems, New York, USA) and exposed
at 4°C for between 24 h and 6 weeks, depending on the probe, developed and counterstained.
The hybridization signal for each brain area was determined using computer-assisted
grain counting software (Zeiss KS 300 3.0, Carl Zeiss Vision, GmbH). For each animal,
silver grains were counted in a fixed circular area over 6 to 10 individual neurons
per subregion. The background, counted over areas of white matter, was subtracted.
Analysis was carried out blind to treatment group.

5-HT transporter (SERT) binding

Serotonin transporter (SERT) binding was determined on brain sections, cut as above,
using (3H)-paroxetine (Perkin Elmer, UK) as previously described [61]. Slides were then exposed to (3H)-sensitive film (Amersham Hyperfilm MP, GE Healthcare, UK) at −80°C for 6 weeks.
Analysis of autoradiographs was performed by measuring the signal over the area of
interest with densitometry software (MCID Basic 7.0, Imaging Research, Inc.). The
background was subtracted.

Statistical analyses

Statistical analyses employed the two-tailed Student’s t test or two-way analysis of variance (ANOVA) with post-hoc analysis using Fisher’s
protected least significant difference test as appropriate, with the exception of
time course of CRFR2 expression, where one-way ANOVA with Dunnett’s post-hoc analysis
was used. Data are presented as mean ± standard error of the mean (SEM). Differences
were considered statistically significant at P < 0.05.

Results

Under basal conditions, where mice were not exposed to stress (other than that caused
by the test itself), CRFR2-null mice and littermate controls showed no differences
in anxiety-related behaviour in two well-validated behavioural tests, the LDT (Figure 2) and the OF test (Figure 3), compared with littermate controls.

Because this finding contrasted with previous reports [50,51], we hypothesized that stressful challenge was required to reveal the role of CRFR2
in anxiety. Another group of mice was tested immediately following 30 min ARS. Again,
no effect of genotype on anxiety-like behaviour was observed (Figures 2 and 3). A further cohort of mice exposed to CVMS was tested 3 to 4 days after the end of
the protocol, to allow for recovery from the final acute stressor, and again no differences
were observed between control and CRFR2 mice in either behavioural test.

However, 3 weeks later, these same CVMS mice were exposed to a single 30-min ARS,
and 24 to 48 h later the CRFR2-null mice showed significantly increased indices of
anxiety compared with controls, with fewer visits to (t = 3.022, P = 0.007, n = 10 or 11), shorter distance travelled in (t = 2.360, P = 0.029, n = 10 or 11), and a trend to less time spent in the light chamber in the LDT (t = 2.062, P = 0.053, n = 10 or 11) (Figure 2), and fewer visits to the centre of (t = 2.271, P = 0.036, n = 10 or 11) and less time spent in (t = 2.231, P = 0.039, n = 10 or 11) the centre and a trend to less time spent in the OF test (t = 1.825, P = 0.085, n = 10 or 11) (Figure 3).

We then examined whether this delayed effect of ARS on anxiety was dependent on prior
CVMS by subjecting a further cohort of mice to ARS alone, and observed the same increased
anxiety-like behaviour 24 to 48 hours post-stress (Figures 2 and 3). In the LDT, CRFR2-null mice spent less time (t = 2.650, P = 0.023, n = 5 to 8) and travelled a shorter distance (t = 2.833, P = 0.016, n = 5 to 8) in the light chamber. In the OF test, CRFR2-null mice spent less time in
(t = 2.675, P = 0.022, n = 5 to 8) and made fewer visits to the centre (t = 3.604, P = 0.004, n = 10 to 11), and travelled a shorter distance (t = 5.078, P = 0.0004, n = 10 to 11).

Serotonergic function is altered in the raphe nuclei of CRFR2-null mice

Following challenge with the 5-HT1AR-specific agonist 8-OH-DPAT, a main effect of treatment (ANOVA: F(1,28) = 4.558, P = 0.044), and an interaction between genotype and treatment was observed in DRN (ANOVA:
F(1,28) = 5.953, P = 0.021) (Figure 4). Post-hoc analysis revealed that controls responded with decreased LCMRglu in both
the DRN (t = 3.235, P = 0.0124, n = 8) and the MRN (t = 2.520, P = 0.047, n = 8) as expected, whereas the raphe nuclei of CRFR2-null mice were unresponsive to
5-HT1AR agonist. Following 5-HT2R-specific agonist DOI challenge, only a main effect of genotype was seen in both
the DRN (ANOVA: F(1,28) = 5.224, P = 0.030) and the MRN (ANOVA: F(1,28) = 5.333, P = 0.029). The pattern of responses was, however, the same as for 8-OH-DPAT.

5-HT responses to stress and 5-HTR agonists are altered in efferent brain regions
of CRFR2-null mice

Following challenge with the 5-HT1AR-specific agonist 8-OH-DPAT, there was a main effect of treatment throughout the
forebrain (ANOVA: F(1,28) = 4.196 for P = 0.05) (Table 1) with a genotype × 8-OH-DPAT interaction observed in some extrapyramidal and limbic
structures. Post-hoc analysis revealed that while controls had decreased LCMRglu in
response to 8-OH-DPAT in extrapyramidal regions as expected, CRFR2-null mice showed
no response. These areas receive projections from the DRN but lack their own 5-HT1AR, indicating that this reflects attenuated DRN response to 5-HT1AR agonist.

Table 1.LCMRglu in efferent brain regions of control and CRFR2-null mice in response to 5-HT1AR or 5-HT2R agonist

In limbic areas, both genotypes decreased LCMRglu significantly (Table 1), but the genotype × 8-OH-DPAT interaction in lateral septum (ANOVA: F(1,28) = 4.654, P = 0.040) and basolateral amygdala (BLA) (ANOVA: F(1,28) = 4.654, P = 0.040) revealed that the CRFR2-null mice had a greater response to 5-HT1AR agonist in these areas. Following DOI challenge, there was again a main effect of
treatment throughout the forebrain (ANOVA: F(1,28) = 4.196 for P = 0.05) (Table 1). Post-hoc analysis revealed that many brain regions showed a significant response
to DOI in CRFR2-null mice but not controls (Table 1), suggesting greater postsynaptic 5-HT2R responsiveness throughout the forebrain in CRFR2-null mice.

We then analyzed 5-HT and 5-HIAA content in the components of an anxiety-related amygdala-subiculum-septal
circuit (Figure 5). There was a main effect of ARS on 5-HT content in the intermediate part of the
lateral septum (LSI) (ANOVA: F(1,22) = 15.41, P = 0.0008) and of genotype on the 5-HIAA:5-HT ratio (ANOVA: F(1,22) = 19.460, P = 0.0002). There was also a genotype × ARS interaction in subiculum on both 5-HT
(ANOVA: F(1,22) = 5.196, P = 0.033) and 5-HIAA:5-HT ratio (ANOVA: F(1,22) = 10.87, P = 0.004), and a main effect of genotype on 5-HIAA:5-HT (ANOVA: F(1,22) = 4.585, P = 0.045).

Serotonergic and corticosteroid receptor gene expression are altered in response to
stress in CRFR2-null mice

To investigate which factors potentially involved in the processes of adaptation to
acute stress might be differentially regulated in CRFR2-null mice as compared with
controls, SERT protein levels (ligand binding) and mRNA levels of serotonergic genes
and corticosteroid receptors (ISH) were quantified in apposite brain nuclei following
ARS or end of CVMS. A time of 12 h post-stress was chosen as appropriate, as altered
expression of these factors at this time has previously been observed by many investigators.
Full results are in Additional file 1; only key significant differences are presented here.

Additional file 1.Serotonergic and corticosteroid gene expression in control and CRFR2-null mice in
response to ARS or CVMS.

In agreement with responses to 8-OH-DPAT, 5-HT1AR mRNA expression did not differ with genotype in the hippocampus or amygdala (Additional
file 1). No effect of genotype or stress was seen in the DRN (Figure 6), but a genotype x stress interaction (ANOVA: F(2,36) = 3.328, P = 0.048) whereby decreased expression in control compared with CRFR2-null mice (t = 2.181, P = 0.036, n = 7) was seen in the MRN following CVMS, and there were trends for ARS to reduce
5-HT1AR expression in CRFR2-null mice (t = 1.702, P = 0.098, n = 6 or 7) but not controls, and for CVMS to reduce 5-HT1AR expression in controls alone (t = 2.020, P = 0.052, n = 6 or 7). There was no appreciable effect of genotype on 5-HT2CR mRNA expression (Additional file 1).

Discussion

This study extends the evidence regarding the importance of CRFR2 in mediating the
processes towards successful behavioural recovery in the period following stress,
and moreover demonstrates that CRFR2 is engaged in the control of serotonergic function
during the same time frame. It further characterizes the stress-sensitive phenotype
of CRFR2-null mice [50,51,62] and reveals fundamental disturbances within components of their serotonergic system.

In contrast with original reports of increased basal levels of anxiety [50,51], in our hands, similar to the findings of Coste et al.[62], CRFR2-null mice do not show increased anxiety-like behaviour compared to controls
until 24 h after exposure to a prior acute stressor. This discrepancy could be due
to differing phenotypes of the three independently generated strains of CRFR2-null
mice, or factors such as age or husbandry. However, the mice in this study are the
same strain as reported with increased anxiety by Bale et al.[50] and an anxious phenotype was described for both group- [50] and singly [51] housed CRFR2-null mice from 9 [40] to 24 [50] weeks of age, but not at 16 weeks [62], meaning that these factors are unlikely to explain the inconsistency. This study
indicates the need for a prior stress for increased anxiogenesis in CRFR2-null mice,
so an alternative explanation is that mice in previous studies might have been inadvertently
previously stressed, for example, by a prior behavioural test. This time course of
the behavioural effects of ARS led us to conclude that CRFR2 has a key role in the
processes leading to behavioural recovery in the hours following exposure to a stressor.

While CRFR2-null mice in our study appear to be in a maladaptive state at 24 hours
following an acute stress, CRFR2-null mice exposed to CVMS are not more anxious than
controls. It could be interpreted from this that CRFR2-null mice have the ability
to cope successfully with this more chronic stress, but it is more likely that both
CRFR2-null mice and controls are affected adversely by CVMS, while CRFR2-null mice
show an exaggerated response to a single acute stressor. Such stressors might release
CRF sufficient to recruit CRFR2 [20], which mediate successful stress coping in normal mice [63]. Alternatively, increased CRFR1 signalling in response to stress might occur; increased
CRF expression in the amygdala and PVN of CRFR2-null mice has been reported [50]. However, the time frame of delayed anxiogenesis in CRFR2-null mice does not correlate
with the expected rapid release of CRF in response to acute stress and its subsequent
negative feedback. The time interval required for CRFR2-null mice to acquire this
anxiety trait suggests that the processes are indirect, and the serotonergic system
is an obvious candidate.

Exogenous CRF administered to the DRN inhibits firing of 5-HT neurons via CRFR1 [15,18], while Ucns or higher levels of CRF increase firing via CRFR2 [32-36]. The raphe nuclei receive inputs from both CRF and Ucn1 neurons [14,15,29], which may therefore regulate serotonergic raphe function physiologically. In support
of this hypothesis, CRFR2-null mice show altered 5-HT/5-HIAA content in the DRN, LSI,
subiculum, CeA and BLA 24 h after ARS, whereas control mice showed a clear change
only in 5-HT content of the CeA. Recent studies of mice with genetically altered Ucn
levels have shown that 5-HT function is dysregulated in these models [39-41] and that CRFR2-null mice show greater sensitivity to elevation of 5-HT levels by
pharmacological means, an observation suggested to be linked to their stress-sensitive
phenotype [64]. Notably, mice deficient in all three Ucns do indeed show a similar phenotype [41] to our observations in CRFR2-null mice, with increased anxiety-like behaviour and
dysregulated activity within 5-HT circuits 24 h following ARS, again evidencing the
importance of CRFR2 here.

Interestingly, CRFR2-null mice show decreased basal neuronal metabolic activity in
the raphe nuclei. This is typically interpreted as evidence of decreased 5-HT firing
activity levels, as while both 5-HT and GABAergic neurons are important functionally
here, GABAergic neurons are present at only 10% of the number of 5-HT neurons [65]. This is an unusual finding under basal conditions in our experience and could be
due to increased raphe 5-HT1AR inhibitory autoreceptor activity, altered 5-HT1AR modulation of raphe GABAergic interneurons that express both CRFR2 and 5-HT1AR, or by inhibition from forebrain postsynaptic receptors including 5-HT1AR and 5-HT2R [66-69]. Increased sensitivity of structures throughout the forebrain to 5-HT2R agonists and to 5-HT1AR in some limbic structures in CRFR2-null mice suggests that postsynaptic receptor
responsiveness is increased, and thus the latter mechanism may be significant. A shift
towards unopposed CRFR1 activity in the raphe nuclei of CRFR2-null mice could also
be a significant factor in mediating these effects or directly inhibiting 5-HT neuronal
activity. Uncontrollable stress, which activates DRN serotonergic neurons [38], is associated with a functional desensitization of 5-HT1AR [70]. We observed no significant differences in 5-HTR expression in the DRN of CRFR2-null
mice and so it is likely that these effects are also mediated by decreased internalization
and desensitization of receptors [71], providing a mechanism for potentially very dynamic responses to stress. Detailed
electrophysiological studies would be required to resolve the mechanism further.

In contrast with forebrain structures, the lack of LCMRglu response in the raphe nuclei
to 5-HTR agonists in CRFR2-null mice suggests tonic inhibition of neuronal activity
here may be close to maximal under basal conditions. The response to 5-HT1AR agonist in extrapyramidal brain areas receiving projections from the DRN [72,73] but lacking their own 5-HT1AR [74,75] was also attenuated. Thus CRFR2 appear to be required for maintaining normal basal
neuronal activity in the raphe nuclei and, in particular, for the balance of 5-HT1AR function here.

CRFR2 are present in both the DRN and the MRN [11,13]. However, studies of stress biology have largely concentrated on the DRN, and so
to relate this altered raphe function to the CRFR2-null behavioural phenotype, we
examined 5-HT responses to stress in the DRN and associated anxiety-related nuclei.
Concentrations of 5-HIAA and the 5-HIAA:5-HT ratio were elevated in CRFR2-null mice
under baseline conditions within the caudal subregion of the DRN (DRC), and these
effects approached significance in the adjacent dorsal subregion (DRD). The DRD and
DRC mediate CRF receptor responses and are considered to be anxiety-related subregions
of the DRN based on anatomical and functional criteria [76,77]. For example, they are activated by anxiogenic drugs [78], CRF-related peptides in vivo[33,79] and in vitro[80], inescapable shock [70], noise stress [80], social defeat [81], the avoidance task on the elevated T-maze [82], acoustic startle [83] and anxiety due to prior experience of intimate partner violence [84]. In support of the specificity of these anxiety-related effects on DRD/DRC serotonergic
systems, in none of these studies were serotonergic neurons in the adjacent ventrolateral
part of the DRN activated. Elevation of 5-HIAA and 5-HIAA:5-HT ratios in the DRC could
be due to an organizational difference in 5-HT systems as a consequence of CRFR2-null
phenotype, or to differential activity that develops later in life. In either case,
DRC neurons appear to have altered baseline activity in adult CRFR2 null mice, which
may reflect a vulnerability to increased anxiety states.

Despite lower 5-HT content in efferent stress-related nuclei under unstressed conditions
in CRFR2-null mice, stress had a greater effect on their 5-HT content at 24 h, in
keeping with their stress-sensitive phenotype. This was particularly evident in LSI,
which receives significant 5-HT projections from the caudal DRN [85,86], in the subiculum and, to a lesser extent, in the CeA. The subiculum is a key structure
in inhibiting the hypothalamic-pituitary-adrenal axis (HPAA) during termination of
the stress response [87] and so altered function here might relate to the higher responsiveness of the HPAA
in CRFR2-null mice following acute stress [50,62].

Not all anxiety-related nuclei examined showed such changes. We found no differences
in the LCMRglu of the bed nucleus of the stria terminalis (BNST) between control and
CRFR2-null mice at either baseline or in response to 8-OH-DPAT or DOI challenge. This
was unexpected, given the pivotal role of the BNST in the control of anxiety states
[88]. However, the serotonergic dysregulation in CRFR2-null mice may be downstream of
the BNST which projects strongly to the DRD/DRC region [89], where CRFR2 are abundant [12,13]. Overexpression of CRF in the BNST induces a decrease in CRFR2 binding selectively
in the DRD/DRC [90] and so it may be that the observed effects in CRFR2-null mice are primarily mediated
here.

5-HT firing activity is generally increased by stress [20] and negative feedback to the DRN ultimately restores balance [66-69,71], as evidenced by essentially unchanged 5-HT and 5-HIAA levels in control mice at
24 h following ARS. It has been previously reported that CRFR2-null mice show greater
sensitivity to 5-HT modulation of stress-induced behaviours [64]. The pattern of increased responses in CRFR2-null mice to both 5-HT1AR and 5-HT2R receptor agonists in areas expressing postsynaptic receptors is in keeping with
this finding. The LSI and BLA are key components of the limbic stress circuitry that
were more responsive to 5-HT1AR agonist in CRFR2-null mice. This might therefore relate to their stress-sensitive
phenotype and indicate a role for these structures in stress recovery.

The MRN has been implicated in mediating a delayed coping response following fear
behaviour induced by CRF in the DRN [35,91]. A delayed increase in 5-HT in the mPFC mediated by CRFR2 in MRN is associated with
cessation of intra-DRN CRF-induced freezing behaviour [91] and is therefore proposed to mediate stress resilience effects [92]. In CRFR2-null mice, the CRFR2-mediated surges in 5-HT neuronal firing from DRN and
MRN cannot occur, and unopposed CRFR1-mediated inhibition in DRN might further contribute
to this [15,18]. The normal 5-HT response in mPFC occurs one to two hours after intra-DRN CRFR2 receptor
activation [35,91], and we infer that the maladaptive state in CRFR2-mice develops after the peak of
this CRFR2-induced increase in mPFC 5-HT and by 24 h after the stress. Thus, we propose
that this delayed activity in efferent 5-HT neurons, which is critical for successful
adaptation to acute stress, is disrupted in CRFR2-null mice. The consequences for
CRFR2-null mice beyond 24 h are unknown, but unlike controls, 5-HT levels at this
time point are increased from basal levels in several limbic nuclei, indicating that
homeostasis has not been restored It is feasible that a lack of negative feedback
within the 5-HT system due to the failure of CRFR2-mediated 5-HT activity might contribute
to this.

Therefore, we propose that a rapid and highly regulated increase in CRFR2 signalling
in response to acute stress, the resultant increase in efferent 5-HT activity and
subsequent negative feedback to restore homeostasis are important for a normal and
successful coping response. The delayed increase in 5-HT in mPMC is of key importance.
Without this orchestrated response, CRFR2-null mice do not respond to stress appropriately,
and there is prolonged anxiety that might account for their well-recognized anxiety
phenotype. This proposed model is presented in Figure 7. There is significant evidence for a role for the MRN in stress recovery [91,92], and this model is consistent with our observation that LCMRglu is lower under basal
conditions in the MRN of CRFR2-null mice while the robust elevation of TPH2 mRNA in
CRFR2-null MRN might be a compensatory response to a lack of CRFR2 activation here.
More detailed analysis of the dynamics of CRF and 5-HT processes in this time frame
and beyond in appropriate subregions of the raphe nuclei, and consideration of the
roles of other mediators of the stress response in CRFR2-null mice, are required to
substantiate this further.

Figure 7.Proposed model for development of prolonged anxiety following acute stress in CRFR2-null
mice. Following acute stress in control mice (A) CRF acting at CRFR1 in the limbic forebrain produces immediate anxiety. High levels
of CRF and potentially Ucn1 activate CRFR1 and more abundant CRFR2 in the DRN with
a net effect to promote early firing of efferent 5-HT neurons to limbic nuclei. Activation
of CRFR2 in the MRN promotes delayed 5-HT release in the mPFC at 1 to 2 h, which acts
at 5-HT1AR to mediate successful coping and anxiolysis by 24 h. Negative feedback
in the 5-HT system restores homeostasis by 24 h. (B) In CRFR2-null mice, the CRFR2-mediated increase in 5-HT firing cannot occur and unopposed
CRFR1 activity might inhibit 5-HT neuronal firing in limbic nuclei even further. Absence
of negative feedback within the 5-HT system contributes to the increased 5-HT levels
observed in limbic areas at 24 h. The temporal dynamics of the 5-HT system following
acute stress are dysregulated and homeostasis has not been restored. Crucially, the
delayed 5-HT activity in mPFC is disrupted and successful coping has not occurred,
resulting in prolonged anxiety.

Owing to the number of mediators involved in stress responses and the complex interactions
among them, other factors in addition to the serotonergic system are likely to be
modified in CRFR2-null mice in the hours following stress exposure, which might have
implications for the longer term. Indeed Ucn1 expression in the Edinger-Westphal nucleus
and CRF in CeA (but not the PVN) are increased in CRFR2 mice [50], which may be a developmental compensatory change that is also responsible at least
in part for their phenotype. Expression of CRFR1 is, however, unaltered. We found
that changes in serotonergic and corticosteroid receptor gene expression in response
to stress were generally greater in CRFR2-null mice, again in keeping with their stress-sensitive
behavioural phenotype. CRFR2-null mice have normal basal HPAA activity, but higher
responsiveness following acute stress [50,62]. Hence, changes such as the observed greater stress-induced increases in hippocampal
SERT levels in CRFR2-null mice may be mediated by glucocorticoids [93,94], adding potentially further complexity to the relationship between CRFR2 and 5-HT
function. Stress also downregulated hippocampal GR mRNA to a greater degree in CRFR2-null
mice, potentially reflecting this expected HPAA hyperactivation. CRFR2-null mice also
had lower basal GR expression in the PVN, possibly reflecting chronically higher HPAA
tone, and CVMS unexpectedly increased this. Discordant regulation of GR expression
in the hippocampus and PVN has been reported previously [95,96], with upregulation of GR by stress suggested to maintain glucocorticoid signalling
to limit HPAA responses during prolonged stress. 5-HT also regulates GR expression,
and this may be mediated through TPH2 activity, in order to regulate HPAA activity
[97]. Both TPH2 and 5-HT1AR mRNAs in MRN were differentially expressed in CRFR2-null mice. TPH2 mRNA levels
in MRN were higher in CRFR2-null mice, and there may be altered afferent control of
DRN activity from here [98], suggesting that the MRN should be more carefully considered in future studies of
CRFR2 function.

Given this proposed role of CRFR2, we might expect expression to be regulated by stress
exposure. We found expression to increase, reaching a maximum at 3 to 12 h post-ARS
and subsequently declining, an effect similar to that seen for CRFR1 when acutely
exposed to ligand [99], while chronic stress decreased CRFR2 mRNA expression in this and a previous study
[100]. Others have observed lower CRFR2 expression in adult rats subjected to maternal
deprivation or in genetically stress-sensitive rodent strains [101,102], suggesting that CRFR2 downregulation has the potential to be permanent in anxious
or stress-sensitive animals. The interesting exceptions are where CRFR2 is increased
by chronically elevated levels of CRF [103] or corticosterone [104], or in a model of maladaptive post-traumatic stress disorder-like behaviour [105]. We hypothesize that while increased CRFR2 activity is required for successful recovery
from stress and subsequent downregulation is a normal adaptive response associated
with healthy coping, that ongoing hyperactivity of CRFR2 might be associated with
a maladaptive stress response. The role of CRFR2 in mediating learned helplessness
in response to uncontrollable stress has implicated CRFR2 activity in the development
of maladaptive behavioural responses [38,79]. However equally, CRFR2 upregulation might be an appropriate secondary adaptation
to a chronic stress. This issue requires further investigation, to assess whether
CRFR2 is a potential target in stress-related psychiatric disorders.

In this study, 5-HT function in the lateral septum and subiculum, sites linked with
anxiety as well as the neural circuitry of reward and addiction [106-108], was particularly altered. Dysregulated serotonergic function has long been linked
to stress-related psychopathologies [109,110] and direct effects of Ucns on CRFR2 in LSI have been observed in rodent models of
these disorders; hence, CRFR2 may play an important role in these processes [111-113].

Conclusions

While the role of CRFR2 in stress recovery was proposed some time ago [63,114], this study provides new information regarding the mechanisms by which this may be
mediated and highlights the importance in the immediate post-stress period. This has
implications for the pathophysiology of psychiatric conditions associated with acute
stress exposure, such as post-traumatic stress disorder, reactive depression and relapse
to substance abuse. As evidence continues to emerge that CRFR2 may mediate its effects
on stress primarily through 5-HT, the potential for involvement in further mood disorders
and ultimately for therapeutic targeting is clear.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

PMJ acquired funding, designed the study, performed experiments, analyzed and interpreted
data and wrote the manuscript. OI designed and performed experiments, analyzed and
interpreted data and prepared figures. RNC, EDP, AN-C and YK performed experiments
and analyzed data. PATK and HJO designed and performed experiments and analyzed and
interpreted data. CAL acquired related funding, designed experiments and analyzed
and interpreted data. JRS and AC acquired related funding and participated in study
design and data interpretation. All authors provided comments and suggestions during
manuscript preparation. All authors read and approved the final manuscript.

Acknowledgements

We are grateful to our funding sources, including Wellcome Trust (grant 083192/Z/07/Z;
PMJ, JRS, AC), the European Research Council (FP7 grant 260463; AC), the National
Institute of Mental Health (NIH Award MH086539; CAL), the Israel Science Foundation
(AC) and the Legacy Heritage Biomedical Science Partnership (AC). The content is solely
the responsibility of the authors and does not necessarily represent the official
views of the National Institute of Mental Health or the National Institutes of Health.